H105A peptide eye drops promote photoreceptor survival in murine and human models of retinal degeneration

Photoreceptor death causes blinding inheritable retinal diseases, such as retinitis pigmentosa (RP). As disease progression often outpaces therapeutic advances, finding effective treatments is urgent. This study focuses on developing a targeted approach by evaluating the efficacy of small peptides derived from pigment epithelium-derived factor (PEDF), known to restrict common cell death pathways associated with retinal diseases. Peptides with affinity for the PEDF receptor, PEDF-R, (17-mer and H105A) delivered via eye drops reached the retina, efficiently promoted photoreceptor survival, and improved retinal function in RP mouse models based on both the rd10 mutation and the rhodopsin P23H mutation. Additionally, intravitreal delivery of AAV-H105A vectors delayed photoreceptor degeneration in the latter RP mouse model. Furthermore, peptide H105A specifically prevented photoreceptor death induced by oxidative stress, a contributing factor to RP progression, in human retinal organoids. This promising approach for peptide eye drop delivery holds significant potential as a therapeutic for preventing photoreceptor death in retinal disorders, offering a high safety profile, low invasiveness and multiple delivery options.


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B.  The highest PS externalization in fundi was at P17 for both models, being higher for rd10 at all ages (Fig. S1B).The fluorescence slightly decreased at P21, to further decrease at P23 and being undetectable at P25.The fluorescence in mutant animals treated with HBSS or in wild type C57Bl/6J mice was undetectable and considered as background controls.To correlate these observations with photoreceptor viability, we compared the height of the outer nuclear layer (ONL) of photoreceptor cells in the retina.The ONL of mice at P15 and P21 showed nuclei that likely corresponded to photoreceptor cells undergoing early apoptosis before their near or complete loss by P25 (Fig. S1C).Depletion of photoreceptors at P25 explained the decrease in PSVue® fluorescence in fundi (shown in Fig. S1B) and precluded use of PSVue® to detect dying cells at P25 for both models.The data agree with previous reports in which in the rd10 and rd10/Serpinf1 -/-mice, loss of photoreceptor phosphodiesterase activity leads to photoreceptor degeneration, starting at postnatal day (P)16, and with near complete loss of photoreceptors by P25 and beyond (13).
Given that cell death is largely regulated by the BCL2 family of interacting proteins, such as BCL2-Associated X protein (BAX) that plays a central role in apoptosis and B-cell lymphoma 2 (BCL2) as an antiapoptosis regulator, we evaluated their distribution in photoreceptors of our mutant mice at P21. Immunofluorescence of retinal cross sections was performed to detect BAX and BCL2.The photoreceptors of both models exhibited BAX, while BCL2 was undetectable (Fig. S1D).Thus, PSVue® fluorescence fundoscopy of mice agreed with detection of photoreceptors undergoing early apoptosis in rd10 and rd10/Serpinf1 -/-mice in vivo, highlighting the age of P21 as endpoint for the rest of the study.
(A) Scheme showing the application of PSVue® 550 delivered via eye drops to rd10 and rd10/Serpinf1 null mice at P16, P20, P22 or P24.Eye drops of 5 µl of a solution of 1 mM PSVue® in HBSS were applied per eye and fluorescence fundoscopy was performed in live animals 24 hours after application.
(B) In vivo detection of PS externalization in photoreceptors using PSVue®.Fluorescence fundoscopy micrographs of retinas at P17, P21, P23 and P25 of rd10, rd10/Serpinf1 -/-, and C57BL/6J mice after 24 h of administering PSVue® are shown.The right image on each row corresponds to retinas with HBSS (vehicle) only at P21, corresponding to background.Quantification of fluorescence intensity was done using ImageJ on images acquired by subtracting the background.Plots were generated using GraphPad.Each data point corresponds to the average of 3 images per retina for a total of 5 retinas per age.Eye drops of H105A peptide solution were administered to rd10 mice either daily (Fig. S2A) or every other day (Fig. S2D).A total of 5 µl of a solution containing H105A peptide at 1 mg/ml in HBSS were applied as eye drops.The vehicle HBSS was added as eye drops in the contralateral eyes.When eye drops were added daily starting at P15 until P24, we noticed a rescue in the ONL thickness in rd10 mice, as shown in Figure S2B.Eye drops also improved rod photoreceptor function in ERG in response to low intensity light at P25 (Fig. S2C).When eye drops of H105A were added every other day, a rescue in the ONL thickness in rd10 mice compared to vehicle was also observed (Fig. S2E), with an improvement in awave of ERG, but no significant effect on the b-wave at P25 (Fig. S2F).The data demonstrate the efficacy of peptide H105A eye drops either daily or every other day in an extended timeline to stabilize photoreceptor morphology and light response function loss in rd10 mice, however, these were less efficacious than daily H105A peptide eye drops.(F) ERG of rd10 mice at P25 and treated as in Fig. S2D were performed in response to a STR.Plots show amplitude (y-axis) for a-and b-wave as function of light intensity (cd/s.m 2 , x-axis).For ERG data shown, the number of mice evaluated were n =5 for rd10 and each data point corresponds to the average ± SD of each genotype by unpaired t-test.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure S3
. H105A expression delivered by AAV2 is maintained at least for 6 months in the transduced retinas.
As from the scheme in figure 5A, each Rho P23H/+ mouse at P5 received AAV-H105A IVT in one eye and AAV-GFP IVT, a GFP expressing control AAV, in the contralateral eye.At P19 and P180, the mRNA and protein were assessed.Figure S4A shows a photograph of the gel with RT-PCR amplicons of H105A mRNA (120 bp) from transduced retinas, confirming the mRNA expression of the virus at P19 and P180.
Fragments migrating as 270 bp correspond to AAV-H105A genomic DNA.Protein was assessed in retinal cross sections by immunofluorescence.Figure S4B shows mouse retinal cross sections with the fluorescence of the expressed GFP in green, DAPI in blue, and immunostaining of rhodopsin, a photoreceptor marker, in red, confirming that the AAV-GFP IVT primarily targeted ganglion cells and Müller glia cells.We also used a custom-generated anti-H105A antibody which was labelled with a FLAGtag, and we validated the specificity of the antibody by several analyses in COS7 cells transduced with AAV-H105A and in sections of AAV-H105A transduced retinas.Figure S4C shows COS7 cells transduced with AAV-H105A and stained with anti-H105A antibodies and DAPI.Transduction of AAV-H105A in COS7 cells resulted in production of the neurotrophic factors as attested by the specific staining of anti-H105A (white arrows) in the cells and not detected with anti-FLAG and Alexa Fluor 568 anti-mouse secondary and tertiary antibodies, respectively (control in the absence of primary anti-H105A antibody).Figure S4D shows that in Rho P23H/+ mouse retinas, AAV2-H105A IVT also resulted in production of H105A, as demonstrated by the specific staining of the anti-H105A (white arrows), that was blocked by competition with an excess of peptide antigen (H105A at 400 nM) and lack of staining with anti-FLAG and Alexa Fluor 568 anti-mouse secondary and tertiary antibodies, respectively.We concluded that a single IVT injection of AAV2-H105A in the Rho P23H/+ mouse produced the neuroprotective agent in the retinal environment at P19 and P180.A PEDF-derived peptide 29-mer (positions 93-121 of human PEDF) delivered by eye drops can prevent the development of experimental dry eye that affects the cornea (56).We designed two peptides 29mer and 29-mer[H105A] with the center of their sequences containing the 17-mer and H105A peptide sequences (in yellow highlight), respectively (Fig. S5A).To evaluate their penetration into the retina and therapeutic effect on mouse photoreceptor cell death, daily eye drops of 5 µl of 29-mer or 29mer[H105A] at 1 mg/ml in HBSS were administered to left eyes and of HBSS (vehicle) to their contralateral right eyes of rd10 and rd10/ Serpinf1 -/-mice starting at P15 (Fig. S5A).At P21 PSVue 550 fluorescence fundoscopy showed no rescue in photoreceptor cell death with either peptide relative to vehicle in rd10 and rd10/Serpinf1 -/-mice (Figs.S5B and S5C).To evaluate the retinal penetration of 29mer and 29-mer[H105A] peptides delivered via eye drops, we designed chemically synthesized peptides conjugated with Alexa Fluor™ 488 dye (Alexa 488) at their amino termini.Solutions of labeled peptides were administered to the left eye of C57Bl/6J mice and HBSS to the right eye.At 1-hour post-delivery, fluorescence fundoscopy showed no fluorescence intensity in the posterior retina for both peptides being similar to the fundi of the eyes that did not received peptide (Fig. S5D).These observations indicated that both 29-mer and 29-mer[H105A] delivered by eye drops were unable to penetrate to the retina and not efficacious in protecting photoreceptors of rd10 mice.For ERG data shown, the number of mice evaluated were n =3 per condition and each data point corresponds to the average ± SD by unpaired t-test.### p < 0.001 and #### p < 0.0001 for statistics between AlexaFl488-H105A and vehicle, and ** p < 0.01 and **** p < 0.0001 for statistics between AlexaFl488-H105A and AlexaFl488-17-mer.We note that eye drops of the labeled peptides AlexaFl-488-17-mer and AlexaFl-488-H105A improved retinal function as determined by ERG, but the labeled AlexaFl-488-R99A did not, as their unlabeled counterparts.
Table S1.Peptides used in the study.

( C )
Retinal cross sections stained with DAPI showing the distribution of nuclei in rd10 and rd10/Serpinf1 - /-mice between P15 and P25.Representative images are shown.The dotted lines demarcate the outer nuclear layer (ONL) for photoreceptor nuclei.(D) Retinal cross sections showing the distribution of BAX and BCL2 in rd10 and rd10/Serpinf1 -/-mice at P21 by immunofluorescence.Representative images are shown.For all immunofluorescence images shown, 5 retinas were used per group.ONL, outer nuclear layer, OS, outer segments, and IS, inner segments.

Figure S2 .
Figure S2.Effects of extending the timeline or administering eye drops of 17-mer[H105A] peptide every other day in rd10 mice.

( A )
Scheme showing a timeline of peptide administration via daily eye drops of H105A peptide to rd10 mice between P15 and P24.Assays were performed at P25. (B) Representative microphotographs of retinal cross sections of rd10 treated as in Fig. S2A and stained with hematoxylin and eosin are shown.To the right, a spider plot illustrating the thickness of the ONL of rd10 mice treated with or without H105A as a function of distance from the optic nerve (ON).For all histology shown, 5 retinas per group were evaluated and each data point corresponds to the average ± SD per location relative to the ON per genotype by unpaired t-test.*p < 0.05, **p < 0.001, ***p < 0.0001, ****p < 0.00001.(C) ERG of rd10 mice at P25 and treated as in Fig. S2A were performed.Plots show amplitude (y-axis) for a-and b-wave as function of light intensity (cd/s.m 2 , x-axis).For ERG data shown, the number of mice evaluated were n =5 for rd10 and each data point corresponds to the average ± SD of each genotype by unpaired t-test.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(D) Scheme showing a timeline of peptide administration via eye drops of H105A peptide to rd10 mice starting at P15 every other day until P23.Assays were at P25. (E) Representative microphotographs of retinal cross sections of rd10 treated as in Fig. S2D and stained with hematoxylin and eosin are shown.To the right, a spider plot illustrating the thickness of the ONL of rd10 mice treated with or without H105A as a function of distance from the optic nerve (ON).For all histology shown, 5 retinas per group were evaluated and each data point corresponds to the average ± SD per location relative to the ON per genotype by unpaired t-test.*p < 0.05, **p < 0.001, ***p < 0.0001, ****p < 0.00001.

( A )
Scheme showing a timeline of daily peptide administration via eye drops to rd10 and rd10/Serpinf1 null mice between P15 and P21.The sequences of the peptides are given, and the yellow highlight corresponds to the 17-mer and 17-mer[H105A] region in the 29-mer and 29-mer[H105A], respectively.(B) Mice were treated with peptides 29-mer and 29-mer[H105A] as indicated in Fig. S5A.Representative fluorescence fundoscopy micrographs of rd10 and rd10/Serpinf1 -/-retinas exposed to PSVue® are shown.Quantification of fluorescence intensity was performed using ImageJ of images acquired by subtracting the background of images of mice treated HBSS without PSVue®.Plots were generated using GraphPad.Each data point corresponds to the average of 3 ROIs per retina for a total of n = 3 retinas per genotype.Scale bar indicates 0.5 mm.(C) AlexaFluor488-labeled 29-mer and AlexaFluor488-29-mer[H105A] were administered to C57Bl/6J mice at P21 days of age via eye drops (5 µl of a solution of each peptide at 1 mg/ml in HBSS per eye).Micrographs of fluorescence fundoscopy performed in vivo of the posterior retinas are shown.Scale bar indicates 0.5 mm.

Figure S5 .
Figure S5.Alexa Fluor 488 labeled 17-mer, H105A and R99A peptides.(A) Standard curves of Alexa Flour-488 labeled peptides in retinal extracts.Plots of fluorescence of each labeled peptide in retinal extracts from C57Bl/6J mouse eyes are shown.Solutions of labeled 17-mer, H105A and R99A peptides at concentrations that ranged between 0.0 µg/ml and 0.0625 µg/ml were prepared.A total volume of 30 µl of each concentration was added to a well.Each data point corresponds to the average of two measurements per concentration.(B) Effect of daily eyedrops of Alexa Fluor-488 labeled peptides on ERG awave and bwave retinal function of rd10 mice.ERGs were performed in rd10 mice treated with eye drops of AlexaFl-488-17mer, AlexaFl-488-H105A and AlexaFl-488-R99A, as shown in the scheme.Plots show amplitude (yaxis) as function of light intensity (cd/s.m 2 , x-axis).For ERG data shown, the number of mice evaluated were n =3 per condition and each data point corresponds to the average ± SD by unpaired t-test.### p < 0.001 and #### p < 0.0001 for statistics between AlexaFl488-H105A and vehicle, and ** p < 0.01 and **** p < 0.0001 for statistics between AlexaFl488-H105A and AlexaFl488-17-mer.We note that eye drops of the labeled peptides AlexaFl-488-17-mer and AlexaFl-488-H105A improved retinal function as determined by ERG, but the labeled AlexaFl-488-R99A did not, as their unlabeled counterparts.